G protein coupled receptors (GPCRs) mediate a large number of physiologic processes. A family of GPCRs that mediates the action of acetylcholine includes five members called muscarinic receptors (M1R-M5R). These receptors are implicated in a number of neurological disorders including depression, Alzheimer's and Parkinson's diseases. The activity and subcellular localization of GPCRs are controlled by a number of intracellular proteins. This proposal concentrates on understanding the regulation of type 3 muscarinic receptor (M3R) by a neurospecific regulator of G protein signaling (RGS), the Gbeta5-RGS7 complex. Recent original findings in this laboratory show that Gbeta5-RGS7 complex can inhibit M3R signaling via a novel mechanism that involves direct binding between M3R and RGS7. M3R activation also causes a dramatic change in subcellular localization of the Gbeta5-RGS7 complex. These phenomena occur in an M3R-selective manner. Furthermore, studies of mice lacking Gbeta5 revealed their similarity to mice lacking M3R. This project will test the hypothesis that the Gbeta5-RGS7 complex regulates M3R signaling via a novel mechanism and/or transfers signals from M3R to intracellular compartments in neurons. Specific Aim 1 will determine structural elements of M3R and RGS7 that are involved in this interaction. Dominant mutants of M3R and RGS7 will be used as molecular tools in Specific Aims 2 and 3. In addition, a series of in vitro assays will be used to study the relationship between the Gbeta5-RGS7 complex and other binding partners of M3R. Aim 2 will study subcellular re-localization of M3R and Gbeta5-RGS7 from the cytosol to endosomal vesicles. The experiments will use advanced imaging methodology, cell fractionation and biochemical approaches to identify subcellular compartments where M3R and Gbeta5-RGS7 re-localize upon receptor activation and search for novel binding partners of this protein complex. Aim 3 will explore the physiologic significance of M3R:Gbeta5-RGS7 interaction in native cells. The experimental design is based on the discovery that Gbeta5 knockout mice have an increased level of epinephrine, which is consistent with high locomotor activity and other changes in these animals. The proposed study will concentrate on the analysis of catecholamine secretion by chromaffin cells of the adrenal medulla from Gbeta5 knockout mice. Experiments will also investigate the neuroendocrine PC12 cell lines with altered expression of Gbeta5- RGS7 and will utilize imaging and biochemical methods. This research will result in better understanding of regulation of neuronal muscarinic acetylcholine receptors, and illuminate novel roles of RGS proteins in regulation of signal transduction and other cellular functions. PUBLIC HEALTH RELEVANCE: This proposal investigates molecular mechanisms that regulate the functions of the neuronal muscarinic acetylcholine receptor type 3. The experiments concentrate on a novel mechanism that involves a neuronal regulator of G protein signaling, RGS7. The proposed experiments will study the function of this protein using biochemical analyses, advanced imaging methods and studies of genetically modified mice. This research will contribute to understanding, at the molecular level, of cognitive, sensory, motor, metabolic and other functions of the nervous system.